Design of Poly(vinyldiaminotriazine)‐Based Nonviral Vectors via Specific Hydrogen Bonding with Nucleic Acid Base Pairs

Abstract

A novel gene transfer system that complexes plasmid DNA (pDNA) through complementary hydrogen bonding of guests with base pairs is reported. Poly(vinyldiaminotriazine) (PVDT) homopolymer and its copolymers with poly(1‐vinyl‐2‐pyrrolidone) are synthesized via conventional radical random polymerization, and water‐soluble fractions are collected. These PVDT‐based polymers efficiently complex pDNA and displace ethidium bromide (EB) in EB‐intercalated pDNA solutions because of the hydrogen‐bonding‐induced constrained state of pDNA. It is also found that upon complexation, pDNA seems to undergo B–C conformation change and circular dichroism spectra assumed a polymer‐and‐salt‐induced (psi)‐type pattern that is rationally ascribed to a certain change in the high‐order structure of DNA condensates. Transmission electron microscopy presented several morphologies of spheres and toroids within aggregates ≥ 100 nm, resembling DNA condensation induced by cationics. In transfection studies using pDNA‐encoding luciferase or enhanced green fluorescent protein, this system could efficiently transfect COS‐1 cells. Compared to the commercial polycation system, ExGen 500, these H‐bonding vectors display several merits such as higher transfection efficiency, lower cytotoxicity, better serum compatibility, and stability in the presence of bovine serum albumin (BSA). The results suggest that PVDT‐based polymers are superior to polycation counterparts with regard to their potential in vivo applications.

Study on cellular internalization of poly(vinyldiaminotriazine)-based hydrogen bonding type non-viral transgene vector

“…To ensure hydrogen bonding interaction, PVDT based polymer/pDNA complexes were made in aqueous solution of pH 6.5, higher than 5.15, the pK a of PVDT (refer to ref. [15] for detailed preparation procedures). Lipofectamine 2000 (Invitrogen) and ExGen 500 (22 kDa linear PEI, Fermentas) were used as positive controls according to manufactures' protocols (Lipofectamine 2000/pDNA and ExGen 500/pDNA ratios were 1 μL/1 μg and 3.3 μL/1 μg, respectively).…”

“…2-vinyl-4,6-diamino-1,3,5-triazine (VDT, Tokyo Kasei Kogyo) and 1-vinyl-2-pyrrolidone (VP, Merck Schuchardt) were used to prepare PVDT homopolymer and PVDT-co-PVP copolymers as described previously [15] .…”

“…Recently, we have developed another type of hydrogen-bonding vectors [15] , that is, poly(2-vinyl-4,6-diamino-1,3,5-triazine)(PVDT) based polymers which is able to recognize nucleic acid bases and their derivative [16,17] and thus complex plasmid DNA (pDNA) via hydrogen bonds between VDT residues and Adenine-Thymine base pairs [18] . In our previous research,…”

“…Then 10 μg of pDNA was mixed with 10 μL YOYO-1 dilution (1 molecule of YOYO-1 per 152 base pairs of pDNA) and incubated for 2 h in the dark. Labeled pDNA was used to make PVDT-based polymer/pDNA complexes based on the protocols in early work [15] . COS-1 cells were seeded in 6-well plates (2×10 5 cells per well) overnight and the medium was replaced with DMEM plus 10% FBS without antibiotics.…”

“…However, SPG cannot complex with dsDNA, so its application in gene therapy was limited. The other clever idea is based on macrocyclic glycocluster, which could effectively mediate gene internalization into Hela and HepG2 cells via saccharidephosphate hydrogen-bonding [12][13][14] .Recently, we have developed another type of hydrogen-bonding vectors [15] , that is, poly(2-vinyl-4,6-diamino-1,3,5-triazine)(PVDT) based polymers which is able to recognize nucleic acid bases and their derivative [16,17] and thus complex plasmid DNA (pDNA) via hydrogen bonds between VDT residues and Adenine-Thymine base pairs [18] . In our previous research,…”